This is a national stage application of international application PCT/EP98/05998, filed Sep. 21, 1998, which in turn claims priority to German application serial no. 197 41 715.9, filed Sep. 22, 1997.
The present invention relates to a pentopyranosyl-nucleoside of the formula (I) or of the formula (II) 
its preparation and use for the production of a therapeutic, diagnostic and/or electronic component.
Pyranosylnucleic acids (p-NAs) are in general structural types which are isomeric to the natural RNA, in which the pentose units are present in the pyranose form and are repetitively linked by phosphodiester groups between the positions C-2xe2x80x2 and C-4xe2x80x2 (FIG. 1). xe2x80x9cNucleobasexe2x80x9d is understood here as meaning the canonical nucleobases A, T, U, C, G, but also the pairs isoguanine/isocytosine and 2,6-diaminopurine/xanthine and, within the meaning of the present invention, also other purines and pyrimidines. p-NAs, namely the p-RNAs derived from ribose, were described for the first time by Eschenmoser et al. (see Pitsch, S et al. Helv. Chim. Acta 1993, 76, 2161; Pitsch, S et al. Helv. Chim Acta 1995, 78, 1621; Angew. Chem. 1996, 108, 1619-1623). They exclusively form so-called Watson-Crick-paired, i.e. purine-pyrimidine- and purine-purine-paired, antiparallel, reversibly xe2x80x9cmeltingxe2x80x9d, quasi-linear and stable duplexes. Homochiral p-RNA strands of the opposite sense of chirality likewise pair controllably and are strictly non-helical in the duplex formed. This specificity, which is valuable for the construction of supramolecular units, is associated with the relatively low flexibility of the ribopyranose phosphate backbone and with the strong inclination of the base plane to the strand axis and the tendency resulting from this for intercatenary base stacking in the resulting duplex and can finally be attributed to the participation of a 2xe2x80x2,4xe2x80x2-cis-disubstituted ribopyranose ring in the construction of the backbone. These significantly better pairing properties make p-NAs pairing systems to be preferred compared with DNA and RNA for use in the construction of supramolecular units. They form a pairing system which is orthogonal to natural nucleic acids, i.e. they do not pair with the DNAs and RNAs occurring in the natural form, which is of importance, in particular, in the diagnostic field.
Eschenmoser et al. (1993, supra) has for the first time prepared a p-RNA, as shown in FIG. 2 and illustrated below.
In this context, a suitable protected nucleobase was reacted with the anomer mixture of the tetrabenzoylribopyranose by action of bis(trimethylsilyl)acetamide and of a Lewis acid such as, for example, trimethylsilyl trifluoromethanesulphonate (analogously to H. Vorbrxc3xcggen, K. Krolikiewicz, B. Bennua, Chem. Ber. 1981, 114, 1234). Under the action of base (NaOH in THF/methanol/water in the case of the purines; saturated ammonia in MeOH in the case of the pyrimidines), the acyl protected groups were removed from the sugar, and the product was protected in the 3xe2x80x2,4xe2x80x2-position under acidic catalysis with p-anisaldehyde dimethyl acetal. The diastereomer mixture was acylated in the 2xe2x80x2-position, and the 3xe2x80x2,4xe2x80x2-methoxybenzylidene-protected 2xe2x80x2-benzoate was deacetalized by acidic treatment, e.g. with trifluoro-acetic acid in methanol, and reacted with dimethoxytrityl chloride. The 2xe2x80x2xe2x86x923xe2x80x2 migration of the benzoate was initiated by treatment with p-nitrophenol/4-(di-methylamino)pyridine/triethylamine/pyridine/n-propanol. Almost all reactions were worked up by column chromatography. The key unit synthesized in this way, the 4xe2x80x2-DMT-3xe2x80x2-benzoyl-1xe2x80x2-nucleobase derivative of the ribopyranose, was then partly phosphitylated and bonded to a solid phase via a linker.
In the following automated oligonucleotide synthesis, the carrier-bonded component in the 4xe2x80x2-position was repeatedly acidically deprotected, a phosphoramidite was coupled on under the action of a coupling reagent, e.g. a tetrazole derivative, still free 4xe2x80x2-oxygen atoms were acetylated and the phosphorus atom was oxidized in order thus to obtain the oligomeric product. The residual protective groups were then removed, and the product was purified and desalted by means of HPLC.
The described process of Eschenmoser et al. (1993, supra), however, shows the following disadvantages:
1. The use of non-anomerically pure tetrabenzoylpentopyranoses (H. G. Fletcher, J. Am. Chem. Soc. 1955, 77, 5337) for the nucleosidation reaction with nucleobases reduces the yields of the final product owing to the necessity of rigorous chromatographic cuts in the following working steps.
2. With five reaction stages, starting from ribopyranoses which have a nucleobase in the 1xe2x80x2-position, up to the protected 3xe2x80x2-benzoates, the synthesis is very protracted and carrying-out on the industrial scale is barely possible. In addition to the high time outlay, the yields of monomer units obtained are low: 29% in the case of the purine unit adenine, 24% in the case of the pyrimidine unit uracil.
3. In the synthesis of the oligonucleotides, 5-(4-nitrophenyl)-1H-tetrazole is employed as a coupling reagent in the automated p-RNA synthesis. The concentration of this reagent in the solution of tetrazole in acetonitrile is in this case so high that the 5-(4-nitrophenyl)-1H-tetrazole regularly crystallizes out in the thin tubing of the synthesizer and the synthesis thus comes to a premature end. Moreover, it was observed that the oligomers were contaminated with 5-(4-nitrophenyl)-1H-tetrazole.
4. The described work-up of p-RNA oligonucleotides, especially the removal of the base-labile protective groups with hydrazine solution, is not always possible if there is a high thymidine fraction in the oligomers.
It was therefore the object of the present invention to make available a novel process for the preparation of pentopyranosylnucleosides by means of which the preparation of known and novel pentopyranosylnucleosides on a larger scale than in known processes is to be made possible and the disadvantages described above are avoided.
A subject of the present invention is therefore a process for the preparation of a pentopyranosylnucleoside, in which, starting from the unprotected pentopyranoside,
(a) in a first step the 2xe2x80x2-, 3xe2x80x2- or 4xe2x80x2-position of the pentopyranoside is first protected, and
(b) in a second step the other position is protected on the 2xe2x80x2-, 3xe2x80x2- or 4xe2x80x2-position.
In a preferred embodiment, a pentopyranosylnucleoside of the formula (I) 
in which
R1 is equal to H, OH, Hal where Hal is equal to Br or Cl, or a radical selected from 
xe2x80x83where i-Pr is equal to isopropyl, R2, R3 and R4 independently of one another, identically or differently, are in each case H, Hal where Hal is equal to Br or Cl, NR5R6, OR7, SR8, xe2x95x90O, CnH2n+1 where n is an integer from 1-12, preferably 1-8, in particular 1-4, a xcex2-eliminable group, preferably a group of the formula xe2x80x94OCH2CH2R18 where R18 is equal to a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) radical, or (CnH2n)NR10R11 where R10R11 is equal to H, CnH2n+1 or R10R11 linked via a radical of the formula 
in which R12, R13, R14 and R15 independently of one another, identically or differently, are in each case H, OR7, where R7 has the abovementioned meaning, or CnH2n+1, or CnH2nxe2x88x921, where n has the abovementioned meaning, and
R5, R6, R7 and R8 independently of one another, identically or differently, is in each case H, CnH2n+1, or CnH2nxe2x88x921, where n has the abovementioned meaning, xe2x80x94C(O)R9 where R9 is equal to a linear or branched, optionally substituted alkyl or aryl radical, preferably a phenyl radical,
X, Y and Z independently of one another, identically or differently, is in each case xe2x95x90Nxe2x80x94, xe2x95x90C(R16)xe2x80x94 or xe2x80x94N(R17)xe2x80x94 where R16 and R17 independently of one another, identically or differently, is in each case H or CnH2n+1 or (CnH2n)NR10R11 having the above-mentioned meanings, the dotted lines represent optional unsaturation, and Sc1 and Sc2 independently of one another, identically or differently, is in each case H or a protective group selected from an acyl, trityl or allyloxycarbonyl group, preferably a benzoyl or 4,4xe2x80x2-dimethoxytrityl (DMT) group,
xe2x80x83or of the formula (II) 
xe2x80x83In which R1xe2x80x2 is equal to H, OH, Hal where Hal is equal to Br or Cl or a radical selected from 
xe2x80x83where i-Pr is equal to isopropyl, R2xe2x80x2, R3xe2x80x2 and R4xe2x80x2 independently of one another, identically or differently, are in each case H, Hal where Hal is equal to Br or Cl, xe2x95x90O, CnH2n+1 or CnH2nxe2x88x921, a xcex2-eliminable group, preferably a group of the formula xe2x80x94OCH2CH2R18 where R18 is equal to a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) radical or (CnH2n)NR10xe2x80x2R11xe2x80x2, where R10xe2x80x2,R11xe2x80x2, independently of one another, has the abovementioned meaning of R10 or R11, and
Xxe2x80x2 is in each case xe2x95x90Nxe2x80x94, xe2x95x90C(R16xe2x80x2)xe2x80x94 or xe2x80x94N(R17xe2x80x2)-, where R16xe2x80x2 and R17xe2x80x2 independently of one another have the abovementioned meaning of R16 or R17, the dotted lines represent optional unsaturation, and Sc1xe2x80x2 and Sc2xe2x80x2 have the above-mentioned meaning of Sc1 and Sc2.
The pentopyranosylnucleoside according to the invention is in general a ribo-, arabino-, lyxo- and/or xylopyranosylnucleoside, preferably a ribopyranosylnucleoside, where the pentopyranosyl moiety can be in the D configuration, but also in the L configuration.
Customarily, the pentopyranosylnucleoside according to the invention is a pentopyranosylpurine, -2,6-diaminopurine, -6-purinethiol, -pyridine, -pyrimidine, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine, -xanthine, -hypoxanthine, -thymidine, -cytosine, -isocytosine, -indole, -tryptamine, -N-phthaloyltryptamine, -uracil, -caffeine, -theobromine, -theophylline, -benzotriazole or -acridine, in particular a pentopyranosylpurine, -pyrimidine, -adenosine, -guanosine, -thymidine, -cytosine, -tryptamine, -N-phthalotryptamine or -uracil.
The compounds also include pentopyranosylnucleosides which can be used as linkers, i.e. as compounds having functional groups which can bond covalently to biomolecules, such as, for example, nucleic acids occurring in their natural form or modified nucleic acids, such as DNA, RNA but also p-NAs, preferably pRNAs. This is surprising, as no linkers are yet known for p-NAs.
For example, these include pentopyranosylnucleosides in which R2, R3, R4, R2xe2x80x2, R3xe2x80x2 and/or Rxe2x80x2 is a 2-phthalimidoethyl or allyloxy radical. Preferred linkers according to the present invention are, for example, uracil-based linkers in which the 5-position of the uracil has preferably been modified, e.g. N-phthaloylaminoethyluracil, but also indole-based linkers, preferably tryptamine derivatives, such as, for example, N-phthaloyltryptamine.
Surprisingly, by means of the present invention more easily handleable pentopyranosyl-N,N-diacylnucleosides, preferably purines, in particular adenosine, guanosine or 6-thioguanosine, are also made available, whose nucleobase can be completely deprotected in a simple manner. The invention therefore also includes pentopyranosylnucleosides, in which R2, R3, R4, R2xe2x80x2, R3xe2x80x2 and/or R4xe2x80x2 is a radical of the formula xe2x80x94N[C(O)R9]2, in particular N6,N6-dibenzoyl-9-(xcex2-D-ribopyranosyl)-adenosine.
It is furthermore surprising that the present invention makes available pentopyranosylnucleosides which carry a protective group, preferably a protective group which can be removed by base or metal catalysis, in particular an acyl group, particularly preferably a benzoyl group, exclusively on the 3xe2x80x2-oxygen atom of the pentopyranoside moiety. These compounds serve, for example, as starting substances for the direct introduction of a further protective group, preferably of an acid- or base-labile protective group, in particular of a trityl group, particularly preferably a dimethoxytrityl group, onto the 4xe2x80x2-oxygen atom of the pentopyranoside moiety without additional steps which reduce the yield, such as, for example, additional purification steps.
Moreover, the present invention makes available pentopyranosylnucleosides which carry a protective group, preferably an acid- or base-labile protective group, in particular a trityl group, particularly preferably a dimethoxytrityl group, exclusively on the 4xe2x80x2-oxygen atom of the pentopyranoside moiety. These compounds too serve, for example, as starting substances for the direct introduction of a further protective group, preferably of a protective group which can be removed by base or metal catalysis, in particular of an acyl group, particularly preferably of a benzoyl group, e.g. on the 2xe2x80x2-oxygen atom of the pentopyranoside moiety, without additional steps which reduce the yield, such as, for example, additional purification steps.
In general, the pentopyranosidenucleosides according to the invention can be reacted in a so-called one-pot reaction, which increases the yields and is therefore particularly advantageous.
The following compounds are preferred examples of the pentopyranosylnucleosides:
A) [2xe2x80x2,4xe2x80x2-Di-O-benzoyl)-xcex2-ribopyranosyl]nucleosides, in particular a [2xe2x80x2,4xe2x80x2-di-O-benzoyl)-xcex2-ribopyranosyl]-adenine, -guanine, -cytosine, -thymidine, -uracil, -xanthine or -hypoxanthine, and an N-benzoyl-2xe2x80x2,4xe2x80x2-di-O-benzoylribopyranosylnucleoside, in particular an -adenine, -guanine or -cytosine, and an N-isobutyroyl-2xe2x80x2,4xe2x80x2-di-O-benzoylribopyranosylnucleoside, in particular an -adenine, -guanine or -cytosine, and an O6-(2-cyanoethyl)-N2-isobutyroyl-2xe2x80x2,4xe2x80x2-di-O-benzoylribopyranosylnucleoside, in particular a -guanine, and an O6(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-2xe2x80x24xe2x80x2-di-O-benzoylribopyranosylnucleoside, in particular a -guanine.
B) xcex2-Ribopyranosylnucleosides, in particular a xcex2-ribopyranosyladenine, -guanine, -cytosine, -thymidine or -uracil, -xanthine or hypoxanthine, and an N-benzoyl-, N-isobutyroyl-, O6-(2-cyanoethyl)- or O6-(2-(4-nitrophenyl)ethyl)-N2-isobutylroyl-xcex2-ribopyranosylnucleoside.
C) 4xe2x80x2-DMT-pentopyranosylnucleosides, preferably a 4xe2x80x2-DMT-ribopyranosylnucleoside, in particular a 4xe2x80x2-DMT-ribopyranosyladenine, -guanine, -cytosine, -thymidine, -uracil, -xanthine or -hypoxanthine, and an N-benzoyl-4xe2x80x2-DMT-ribopyranosylnucleoside, in particular an N-benzoyl-4xe2x80x2-DMT-ribopyranosyladenine, -guanine or -cytosine, and an N-isobutyroyl-4xe2x80x2-DMT-ribopyranosylnucleoside, in particular N-isobutyroyl-4xe2x80x2-DMT-ribopyranosyladenine, -guanine or -cytosine and an O6-(2-cyanoethyl)-N2-isobutyroyl-4xe2x80x2-DMT-ribopyranosylnucleoside, in particular an O6-(2-cyanoethyl)-N2-isobutyroyl-4xe2x80x2-DMT-ribopyranosylguanine, and an O6-(2-(-4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-ribopyranosylnucleoside, in particular an O6-(2-(-4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-ribopyranosylguanine.
D) xcex2-Ribopyranosyl-N,Nxe2x80x2-dibenzoyladenosine or xcex2-ribopyranosyl-N,Nxe2x80x2-dibenzoylguanosine.
Suitable precursors for the oligonucleotide synthesis are, for example, 4xe2x80x2-DMT-pentopyranosylnucleoside-2xe2x80x2-phosphitamide/-H-phosphonate, preferably a 4xe2x80x2DMT-ribopyranosylnucleoside-2xe2x80x2-phosphitamide/-H-phosphonate, in particular a 4xe2x80x2-DMT-ribopyranosyladenine-, -guanine-, -cytosine-, -thymidine-, -xanthine-, hypoxanthine-, or -uracil-2xe2x80x2-phosphitamide/-H-phosphonate and an N-benzoyl-4xe2x80x2-DMT-ribopyranosyladenine-, -guanine- or -cytosine-2xe2x80x2-phosphitamide/-H-phosphonate and an N-isobutylroyl-4xe2x80x2-DMT-ribopyranosyladenine-[sic], -guanine- or -cytosine-2xe2x80x2-phosphitamide/-H-phosphonate, O6(2-cyano-ethyl)-4xe2x80x2-DMT-ribopyranosylguanine-, -xanthine-, -hypoxanthine-2xe2x80x2-phosphitamide/-H-phosphonate or O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-ribopyranosylguanine, and for the coupling to the solid carrier, for example, 4xe2x80x2-DMT-pentopyranosylnucleoside-2xe2x80x2-succinate, preferably a 4xe2x80x2-DMT-ribopyranosylnucleoside-2xe2x80x2-succinate, in particular a 4xe2x80x2DMT-ribopyranosyladenine-, -guanine-, -cytosine-, thymidine-, -xanthine-, -hypoxanthine- or -uracil-2xe2x80x2-succinate and an N-benzoyl-4xe2x80x2-DMT-ribopyranosyl-adenine-, -guanine- or -cytonsine-2xe2x80x2-succinate [sic] and an N-isobutyroyl-4xe2x80x2-DMT-ribopyranosyladenine-, -guanine- or -cytosine-2xe2x80x2-succinate, O-(2-cyanoethyl)-4xe2x80x2-DMT-ribopyranosylguanine-2xe2x80x2-succinate and an O6-(2-(4-nitrophenyl)ethyl)-N2-isobutyroyl-4xe2x80x2-DMT-ribopyranosylguanine-2xe2x80x2-succinate.
The process according to the invention is not restricted to the nucleobases described in the cited literature, but can surprisingly be carried out successfully using a large number of natural and synthetic nucleobases. Moreover, it is particularly surprising that the process according to the invention can be carried out in high yields and with a time saving of.on average 60% in comparison with the process known from the literature, which is particularly advantageous for industrial application. In addition, using the process according to the invention the purification steps necessary in the process described in the literature, e.g. chromatographic intermediate purifications, are not necessary and the reactions can in some cases be carried out as a so-called one-pot reaction, which markedly increases the space/time yields.
In a particular embodiment, in the case of a 2xe2x80x2-protected position a rearrangement of the protective group from the 2xe2x80x2-position to the 3xe2x80x2-position takes place, which in general is carried out in the presence of a base, in particular in the presence of N-ethyldiisopropylamine and/or triethylamine. According to the present invention, this reaction can be carried out particularly advantageously in the same reaction container as the one-pot reaction.
In a further preferred embodiment, the pyranosylnucleoside is protected by a protective group Sc1, Sc2, Sc1xe2x80x2 or Sc2xe2x80x2 which is acid-labile, base-labile or can be removed with metal catalysis, the protective groups Sc1 and Sc1xe2x80x2 preferably being different from the protective groups Sc2 and Sc2xe2x80x2.
In general, the protective groups mentioned are an acyl group, preferably an acetyl, benzoyl, nitrobenzoyl and/or methoxybenzoyl group, trityl groups, preferably a 4,4xe2x80x2-dimethoxytrityl (DMT) group or a xcex2-eliminable group, preferably a group of the formula OCH2CH2R18 where R18 is equal to a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl (Fmoc) group.
It is particularly preferred if the 2xe2x80x2- or 3xe2x80x2-position is protected by a protective group which is base-labile or can be removed with metal catalysis, preferably by an acyl group, in particular by an acetyl, benzoyl, nitrobenzoyl and/or methoxybenzoyl group, and/or the 4xe2x80x2-position is protected by an acid- or base-labile protective group, preferably by a trityl and/or Fmoc group, in particular by a DMT group.
Unlike the process known from the literature, the process according to the invention consequently manages without acetal protective groups, such as acetals or ketals, which avoids additional chromatographic intermediate purifications and consequently allows the reactions to be carried out as one-pot reactions with surprisingly high space/time yields.
The protective groups mentioned are preferably introduced at low temperatures, as by this means they can be introduced surprisingly selectively.
Thus, for example, the introduction of a benzoyl group takes place by reaction with benzoyl chloride in pyridine or in a pyridine/methylene chloride mixture at low temperatures. A DMT group can be introduced, for example, by reaction with DMTCl in the presence of a base, e.g. of N-ethyldiisopropylamine (Hxc3xcnig""s base), and, for example, of pyridine, methylene chloride or a pyridine/methylene chloride mixture at room temperature.
It is also advantageous if after the acylation and/or after the rearrangement of the 2xe2x80x2- to the 3xe2x80x2-position which is optionally carried out, the reaction products are purified by chromatography. Purification after the tritylation is not necessary according to the process according to the invention, which is particularly advantageous.
The final product, if necessary, can additionally be further purified by crystallization.
Another subject of the present invention is a process for the preparation of a ribopyranosylnucleoside, in which
(a) a protected nucleobase is reacted with a protected ribopyranose,
(b) the protective groups are removed from the ribopyranosyl moiety of the product from step (a), and
(c) the product from step (b) is reacted according to the process described above in greater detail.
In this connection, in order to avoid further time- and material-consuming chromatography steps, it is advantageous only to employ anomerically pure protected pentopyranoses, such as, for example, tetrabenzoylpentopyranoses, preferably xcex2-tetrabenzoylribopyranoses (R. Jeanloz, J. Am. Chem. Soc. 1948, 70, 4052).
In a further embodiment, a linker according to formula (II), in which R4xe2x80x2 is (CnH2n)NR10xe2x80x2R11xe2x80x2 and R10xe2x80x2R11xe2x80x2 is linked by means of a radical of the formula (III) having the meaning already designated, is advantageously prepared by the following process:
(a) a compound of the formula (II) where R4xe2x80x2 is equal to (CnH2n)OSc3 or (CnH2n)Hal, in which n has the above-mentioned meaning, Sc3 is a protective group, preferably a mesylate group, and Hal is chlorine or bromine, is reacted with an azide, preferably in DMF, then
(b) the reaction product from (a), is preferably reduced with triphenylphosphine, e.g. in pyridine, then
(c) the reaction product from (b) is reacted with an appropriate phthalimide, e.g. N-ethoxycarbonylphthalimide, and
(d) the reaction product from (c) is reacted with an appropriate protected pyranose, e.g. ribose tetrabenzoate, and finally
(e) the protected groups are removed, for example with methylate, and
(f) the further steps are carried out as already described above.
In addition, indole derivatives as linkers have the advantage of the ability to fluoresce and are therefore particularly preferred for nanotechnology applications in which it may be a matter of detecting very small amounts of substance. Thus indole-1-ribosides have already been described in N. N. Suvorov et al., Biol. Aktivn. Soedin., Akad. Nauk SSSR 1965, 60 and Tetrahedron 1967, 23, 4653. However, there is no analogous process for preparing 3-substituted derivatives. In general, their preparation takes place via the formation of an aminal of the unprotected sugar component and an indoline, which is then converted into the indole-1-riboside by oxidation. For example, indole-1-glucosides and -1-arabinosides have been described (Y. V. Dobriynin et al. Khim.-Farm Zh. 1978, 12, 33), whose 3-substituted derivatives were usually prepared by means of Vielsmeier""s reaction. This route for the introduction of aminoethyl units into the 3-position of the indole is too complicated, however, for industrial application.
In a further preferred embodiment, a linker according to formula (I), in which X and Y independently of one another, identically or differently, are in each case xe2x95x90C(R16) where R16 is equal to H or CnH2n and Z xe2x95x90C(R16)xe2x80x94 where R16 is equal to (CnH2n)NR10R11 is therefore advantageously prepared by the following process:
(a), the appropriate indoline, e.g. N-phthaloyltryptamine, is reacted with a pyranose, e.g. D-ribose, to give the nucleoside triol, then
(b) the hydroxyl groups of the pyranosyl moiety of the product from (a) are preferably protected with acyl groups, e.g. by means of acetic anhydride, then
(c) the product from (b) is oxidized, e.g. by 2,3-dichloro-5,6-dicyanoparaquinone, and
(d) the hydroxyl protective groups of the pyranosyl moiety of the product from (c) are removed, for example, by means of methylate and finally
(e) the further steps as already described above are carried out.
This process, however, cannot only be used in the case of ribopyranoses, but also in the case of ribofuranoses and 2xe2x80x2-deoxyribofuranoses or 2xe2x80x2-deoxyribopyranoses, which is particularly advantageous. The nucleosidation partner of the sugars used is preferably tryptamine, in particular N-acyl derivatives of tryptamine, especially N-phthaloyltryptamine.
In a further embodiment, the 4xe2x80x2-protected, preferably, the 3xe2x80x2,4xe2x80x2-protected pentopyranosylnucleosides are phosphitylated in a further step or bonded to a solid phase.
Phosphitylation is carried out, for example, by means of monoallyl N-diisopropylchlorophosphoramidite in the presence of a base, e.g. N-ethyldiisopropylamine or by means of phosphorus trichloride and imidazole or tetrazole and subsequent hydrolysis with the addition of a base. In the first case, the product is a phosphoramidite and in the second case an H-phosphonate. The bonding of a protected pentopyranosylnucleoside according to the invention to a solid phase, e.g. xe2x80x9clong-chain alkylamino-controlled pore glassxe2x80x9d (CPG, Sigma Chemie, Munich) can be carried out, for example, as described in Eschenmoser et al. (1993).
The compounds obtained serve, for example, for the preparation of pentopyranosylnucleic acids.
A further subject of the present invention is therefore a process for the preparation of a pentopyranosylnucleic acid, having the following steps:
(a) in a first step a protected pentopyranosylnucleoside is bonded to a solid phase as already described above and
(b) in a second step the 3xe2x80x2-,4xe2x80x2-protected pentopyranosylnucleoside bonded to a solid phase according to step (a) is lengthened by a phosphitylated 3xe2x80x2-, 4xe2x80x2-protected pentopyranosylnucleoside and then oxidized, for example, by an aqueous iodine solution, and
(c) step (b) is repeated with identical or different phosphitylated 3xe2x80x2-,4xe2x80x2-protected pentopyranosylnucleosides until the desired pentopyranosylnucleic acid is present.
Acidic activators such as pyridinium hydrochloride, particularly benzimidazolium triflate, are suitable as a coupling reagent when phosphoramidites are employed, preferably after recrystallizing in acetonitrile and after dissolving in acetonitrile, as in contrast to 5-(4-nitrophenyl)-1H-tetrazole as a coupling reagent no blockage of the coupling reagent lines and contamination of the product takes place.
Arylsulphonyl chlorides, diphenyl chlorophosphate, pivaloyl chloride or adamantoyl chloride are [sic] particularly suitable as a coupling reagent when H-phosphonates are employed.
Furthermore, it is advantageous by means of addition of a salt, such as sodium chloride, to the protective-group-removing hydrazinolysis of oligonucleotides, in particular of p-NAs, preferably of p-RNAs, to protect pyrimidine bases, especially uracil and thymine, from ring-opening, which would destroy the oligonucleotide. Allyloxy groups can preferably be removed by palladium [Pd(0)] complexes, e.g. before hyrazinolysis.
In a further particular embodiment, pentofuranosylnucleosides, e.g. adenosine, guanosine, cytidine, thymidine and/or uracil occurring in their natural form, can also be incorporated in step (a) and/or step (b), which leads, for example, to a mixed p-NA-DNA or p-NA-RNA.
In another particular embodiment, in a further step an allyloxy linker of the formula
Sc4NH(CnH2n)CH(OPSc5Sc6)CnH2nOSc7 xe2x80x83xe2x80x83(IV)
in which Sc4 and Sc7 independently of one another, identically or differently, are in each case a protective group in particular selected from Fmoc and/or DMT,
Sc5 and Sc6 independently of one another, identically or differently, are in each case an allyloxy and/or diisopropylamino group, can be incorporated. n has the meaning already mentioned above.
A particularly preferred allyloxy linker is (2-(S)-N-Fmoc-O1-DMT-O2-allyloxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol).
Starting from, for example, lysine, in a few reaction steps amino-terminal linkers can thus be synthesized which carry both an activatable phosphorus compound and an acid-labile protective group, such as DMT, and can therefore easily be used in automatable oligonucleotide synthesis (see, for example, P. S. Nelson et al., Nucleic Acid Res. 1989, 17, 7179; L. J. Arnold et al., WO 8902439). The repertoire was extended in the present invention by means of a lysine-based linker, in which instead of the otherwise customary cyanoethyl group on the phosphorus atom an allyloxy group was introduced, and which can therefore be advantageously employed in the Noyori oligonucleotide method (R. Noyori, J. Am. Chem. Soc. 1990, 112, 1691-6).
p-NAs and in particular the p-RNAs form stable duplexes with one another and in general do not pair with the DNAs and RNAs occurring in their natural form. This property makes p-NAs preferred pairing systems.
Such pairing systems are supramolecular systems of non-covalent interaction, which are distinguished by selectivity, stability and reversibility, and whose properties are preferably influenced thermodynamically, i.e. by temperature, pH and concentration. Such pairing systems can also be used, for example, on account of their selective properties as xe2x80x9cmolecular adhesivexe2x80x9d for the bringing together of different metal clusters to give cluster associates having potentially novel properties [see, for example, R. L. Letsinger et al., Nature 1996, 382, 607-9; P. G. Schultz et al., Nature 1996, 382, 609-11]. Consequently, the p-NAs are also suitable for use in the field of nanotechnology, for example for the preparation of novel materials, diagnostics and therapeutics and also microelectronic, photonic or optoelectronic components and for the controlled bringing together of molecular species to give supramolecular units, such as, for example, for the (combinatorial) synthesis of protein assemblies [see, for example, A. Lombardi, J. W. Bryson, W. F. DeGrado, Biomolekxc3xcls (Pept. Sci.) 1997, 40, 495-504], since p-NAs form pairing systems which are strongly and thermodynamically controllable. A further application therefore arises, especially in the diagnostic and drug discovery field, due to the possibility of providing functional, preferably biological units such as proteins or DNA/RNA sections with a p-NA code which does not interfere with the natural nucleic acids (see, for example, WO93/20242).
A biomolecule, e.g. DNA or RNA, can be used for non-covalent linking with.another biomolecule, e.g. DNA or RNA, if both biomolecules contain sections which, as a result of complementary sequences of nucleobases, can bind to one another by formation of hydrogen bridges. Biomolecules of this type are used, for example, in analytical systems for signal amplification, where a DNA molecule whose sequence is to be analysed is on the one hand to be immobilized by means of such a non-covalent DNA linker on a solid support, and on the other hand is to be bonded to a signal-amplifying branched DNA molecule (bDNA) (see, for example, S. Urdea, Biol/Technol. 1994, 12, 926 or U.S. Pat. No. 5,624,802). An essential disadvantage of the last-described systems is that to date they are subject with respect to sensitivity to the processes for nucleic acid diagnosis by polymerase chain reaction (PCR) (K. Mullis, Methods Enzymol. 1987, 155, 335). This is to be attributed, inter alia, to the fact that the non-covalent bonding of the solid support to the DNA molecule to be analysed as well as the non-covalent bonding of the DNA molecule to be analysed does not always take place specifically, as a result of which a mixing of the functions xe2x80x9csequence recognitionxe2x80x9d and xe2x80x9cnon-covalent bondingxe2x80x9d occurs. The use of p-NAs as an orthogonal pairing system which does not intervene in the DNA or RNA pairing process solves this problem advantageously, as a result of which the sensitivity of the analytical processes described can be markedly increased.
The pentopyranosylnuclosides or pentopyranosylnucleic acids prepared according to the process according to the invention are therefore suitable for the production of a medicament, such as, for example, of a therapeutic, diagnostic and/or electronic component, for example in.the form of a conjugate, i.e. in combination with a biomolecule.
Conjugates within the meaning of the present invention are covalently bonded hybrids of p-NAs and other biomolecules, preferably a peptide, protein or a nucleic acid, for example an antibody or a functional moiety thereof or a DNA and/or RNA occurring in its natural form. Functional moieties of antibodies are, for example, Fv fragments (Skerra and Plutckthun (1988) Science 240, 1038), single-chain Fv fragments (scFv; Bird et al. (1988), Science 242, 423; Huston et al. (1988) Proc. Natl. Acad. Sci. USA, 85, 5879) or Fab fragments (Better et al. (1988) Science 240, 1041).
Biomolecule within the meaning of the present invention is understood as meaning a naturally occurring substance or a substance derived from a naturally occurring substance.
In a preferred embodiment, they are in this case p-RNA/DNA or p-RNA/RNA conjugates.
Conjugates are preferably used when the functions xe2x80x9csequence recognitionxe2x80x9d and xe2x80x9cnon-covalent bondingxe2x80x9d must be realized in a molecule, since the conjugates according to the invention contain two pairing systems which are orthogonal to one another.
Both sequential and convergent processes are suitable for the preparation of conjugates.
In a sequential process, for example after automated synthesis of a p-RNA oligomer has taken place directly on the same synthesizerxe2x80x94after readjustment of the reagents and of the coupling protocolxe2x80x94a DNA oligonucleotide, for example, is additionally synthesized. This process can also be carried out in the reverse sequence.
In a convergent process, for example, p-RNA oligomers having amino-terminal linkers and, for example, DNA oligomers having, for example, thiol linkers are synthesized in separate operations. An iodoacetylation of the p-RNA oligomer and the coupling of the two units according to protocols known from the literature (T. Zhu et al., Bioconjug. Chem. 1994, 5, 312) is then preferably carried out.
Convergent processes prove to be particularly preferred on account of their flexibility.
The term conjugate within the meaning of the present invention is also understood as meaning so-called arrays. Arrays are arrangements of immobilized recognition species which, especially in analysis and diagnosis, play an important role in the simultaneous determination of analytes. Examples are peptide arrays (Fodor et al., Nature 1993, 364, 555) and nucleic acid arrays (Southern et al. Genomics 1992, 13, 1008; Heller, U.S. Pat. No. 5,632,957). A higher flexibility of these arrays can be achieved by binding the recognition species to coding oligonucleotides and the associated, complementary strands to certain positions on a solid carrier. By applying the coded recognition species to the xe2x80x9canti-codedxe2x80x9d solid carrier and adjustment of hybridization conditions, the recognition species are non-covalently bonded to the desired positions. As a result, various types of recognition species, such as, for example, DNA sections, antibodies, can only be arranged simultaneously on a solid carrier by use of hybridization conditions (see FIG. 3). As a prerequisite for this, however, codons and anticodons are necessary which are extremely strong and selectivexe2x80x94in order to keep the coding sections as short as possiblexe2x80x94and do not interfere with natural nucleic acid. p-NAs, preferably p-RNAs, are particularly advantageously suitable for this.
The term xe2x80x9ccarrierxe2x80x9d within the meaning of the present invention is understood as meaning material, in particular chip material, which is present in solid or alternatively gelatinous form. Suitable carrier materials are, for example, ceramic, metal, in particular noble metal, glasses, plastics, crystalline materials or thin layers of the carrier, in particular of the materials mentioned, or (bio)molecular filaments such as cellulose, structural proteins.
The present invention therefore also relates to the use of pentopyranosylnucleic acids, preferably ribopyranosylnucleic acids, for encoding recognition species, preferably natural DNA or RNA strands or proteins, in particular antibodies or functional moieties of antibodies. These can then be hybridized with the appropriate codons on a solid carrier according to FIG. 4. Thus arrays which are novel and diagnostically useful can always be built up in the desired positions on a solid carrier which is equipped with codons in the form of an array only by adjustment of hybridization conditions using combinations of recognition species which are always novel. If the analyte, for example a biological sample such as serum or the like, is then applied, the species to be detected are bonded to the array in a certain pattern which is then recorded indirectly (e.g. by fluorescence labelling of the recognition species) or directly (e.g. by impedance measurement at the linkage point of the codon). The hybridization is then eliminated by suitable conditions (temperature, salts, solvents, electrophoretic processes) so that again only the carrier having the codons remains. This is then again loaded with other recognition species and is used, for example, for the same analyte for the determination of another sample. The always new arrangement of recognition species in the array format and the use of p-NAs as pairing systems is particularly advantageous compared with other systems, see, for example, WO 96/13522 (see 16, below).
A further subject of the present invention therefore also relates in particular to a diagnostic comprising a pentopyranosylnucleoside described above or a conjugate according to the invention, as already described above in greater detail.